幽门螺杆菌过氧化氢酶脂质体疫苗免疫预防研究

目的探讨制备脂质体包裹重组幽门螺杆菌过氧化氢酶(Kat)口服疫苗的方法,并用Hp感染的小鼠模型评价其在预防Hp感染中的作用。方法将PET-22(+)/Kat在BL21(DE3)大肠杆菌表达,表达Kat的包涵体经洗涤、变性、复性,采用Q Sepharose Fast Flow阴离子交换层析和Sephacryl S-200凝胶过滤层析分离纯化Kat重组蛋白,用薄膜分散法制备以卵磷脂和胆固醇为膜组分包裹的Kat重组蛋白口服疫苗,并用透射电镜测定其粒径。BALB/c小鼠分为5组,分别通过灌胃方法给予PBS、空白脂质体、Kat重组蛋白+CT、脂质体包裹Kat重组蛋白、脂质体包裹Kat重组蛋白和CT,每周1次共4次,末次攻击2周再用活Hp攻击3次,3周后处死小鼠,行胃组织快速尿素酶试验、Hp的定植半定量、炎症程度及其炎症活动度的评分。结果以包涵体为主的表达产物占全菌总蛋白的24.4%,经纯化获得纯度为95%的重组蛋白,制备的脂质体粒径为0.7±0.4μm。PBS组和空白脂质体组保护率均为0,而Kat重组蛋白+CT组、脂质体包裹Kat重组蛋白组、脂质体包裹Kat重组蛋白和CT组的保护率分别为73.3%、66.7%和86.7%,且均能使免疫小鼠胃粘膜Hp感染数目明显减少,炎症反应减轻。结论口服脂质体部能分代替免疫佐剂的作用,将其作为Hp疫苗的免疫佐剂,具有广泛的应用前景。

Immune prophylaxia of the liposome-encapsulated catalase protein vaccine for Helicobacter pylori infections

To prepare the oral liposome-encapsulated vaccine with the recombinant catalase protein(Kat) of Helicobacter pylori(Hp) and to investigate its effect in a Hp-infected mouse model,the recombinant vector pET-22(+)/Kat was transformed into E.coli BL21(DE3),and the inclusion bodies of Kat were washed,denatured and renatured.The two-step chromatograohic procedure(Q Sapharose fast flow exchange and Sephacry S-200 gel exclusion) were used for purification.The oral liposome-encapsulated catalase protein vaccine with phosphatidylm choline(PC) and cholesterol was prepared by using the film method,whose size distribution of folate liposome was measured by electron microscopy.BALB/c mice were divided into 5 groups,in which each group received the inoculations of PBS alone,liposome alone,Kat plus cholera toxin(CT),liposome-encapsulated Kat and liposome-encapsulated Kat plus CT respectively by oral route once a week for 4 weeks.After the last immunization, all mice were challenged with live Hp 3 times at two weeks interval and were sacrificed 3 weeks after the last challenge.The activities of Hp were determined by rapid urease test,semi-quantization of the bacterial colonization density and the grades of the inflammation severity and activity of the gastric histopathology.The experimental result revealed that the expressed product of inclusion body accounted to 24.4% of the total bacterial proteins,and the purity of the recombinant was about 95% after purification.After immunization,the protective rates of the groups with Kat plus CT,liposome-encapsulated Kat and liposome-encapsulated Kat plus CT were 73.3%,66.7% and 86.7% respectively,while those of the PBS or liposome alone were all 0%.The incidences of gastric mucosa infection with Hp were significant decreased in the mice inoculated with the above mentioned three immune antigens.It is clear that the oral liposome is a potential immunization adjuvant candidate for Hp vaccine and can be used in the prevention and eradiation of the Hp infections.